with cytochalasin D at 10 ；g/ml (Cyto D, ;) or with DMSO as control (Ctr, ;) for 30 min.

Firefly luciferase was then added at 49 ；g/ml to each sample for 30 min. After extensive

washes with PBS, cells were lysed in 100 ；l of 1% CE in TBS, pH 7.4. After centrifugation, 129

20 ；l of supernatants were analyzed for luciferase activity with a luminometer. The graph shows the mean value (? SD) of triplicate samples. Results are representative of at least three independent experiments.

Supplemental Figure 3

ExoA does not affect Firefly luciferase uptake or in vitro refolding. A. PBS control (;) or

ExoA (;) at 330 ；g/ml was internalized together with Firefly luciferase for 15 min at 37ºC. After extensive washing with PBS, cells were lysed in 100 ；l of 1% CE in TBS, pH 7.4. After 129

1

centrifugation, 20 ；l of supernatants were analyzed for luciferase activity with a luminometer. The graph shows the percent of luciferase activity compared to control cells (;) (? SD) of

triplicate samples. Results are representative of at least three independent experiments. B. Cells

left untreated (;) or that had internalized ExoA (;) or BSA (;) at 330 ；g/ml for 15 min at 37ºC

were permeabilized with SLO as described in Materials and Methods. After centrifugation at 14,000 g for 5 min at 4ºC, supernatants were used for in vitro refolding of chemically unfolded

Firefly luciferase as described in Materials and Methods. 20 ；l of refolding reactions were

analyzed for luciferase activity with a luminometer; refolding in DPBS buffer alone was used as a control. The graph shows the mean value (? SD) of triplicate samples. Results are representative of at least three independent experiments.

Supplemental Figure 4

Denaturation by guanidinium chloride followed by dialysis results in soluble inactive Renilla reniformis luciferase. The enzyme was chemically unfolded in 6M Guanidine-HCl in

ICT buffer for 2 hrs and then dialyzed overnight against PBS. Luciferase activities of 0.1 ngof

folded (;) or unfolded (;) enzyme in PBS were measured with a luminometer. Lack of

KG-1 cells were resuspended in 1.5 ml 1% FBS IMDM. Lucifer Yellow at the final concentration of 250 ；g/ml was added for the indicated time points (solid lines). After extensive washes, Lucifer Yellow fluid phase uptake was evaluated by flow cytometric analysis. Incubation performed at 4ºC (dashed lines) served as a control.